BIOTECHNOLOGY LEARNING CURVES SERIES: Introduction to Vaccine Production (Part 7)

Bacterial Vaccines: Preparation of Inoculum Media (Tryptic Soil Broth)

The inoculums media used for the fermentation process is called Triptych Soil Broth (TSB). 15 L TSB media is used to inoculate into 200 L fermentation media. To produce 1 L of TSB solution, 30g of TSB powder is diluted with 1L water. The figures above show the process of making the TSB inoculums media.

After that, the colonies of bacteria will be transfered to the 15L inoculum TSB media and incubated for 24 hours. This step is to enable the bacteria to further propagate before being transfered into the bioreactor.

BIOTECHNOLOGY LEARNING CURVES SERIES: Introduction to Vaccines Production (Part 6)

Bacterial Vaccines: Preparation of Seed Bacteria

Eight Pasteurella multocida strains which have different antigenic structures seed are taken out from the freezer and allow standing for half an hour to thaw the seed. Then, it was sub cultured on the blood agar and incubated at 37^oC for 24 hours. Due to the rich nutrient provided from the blood agar, there should be a good growth of the bacteria. The purity can be observed directly according to the characteristic, morphology and smell. If it found out contains the contaminant, another sub culture is done to further purify the bacteria. After that, 20 pure single colonies from the plate are inoculated into the universal bottles containing TSB and incubated at 37^oC for 4 hours.

BIOTECHNOLOGY LEARNING CURVES SERIES: Introduction to Vaccine Production (Part 5)

Viral Vaccine: Eggs Drilling, Virus Inoculation and Vaccine Harvesting

Before proceeding to the inoculation process, the eggs have to be drilled to make a tiny hole in order to let the syringe penetrate the egg shell. A small electric rotary (driller) is used to drill a tiny hole on the drilling point that was marked during the marking process. The hole is made by slowly touch the rotary part of the driller on the drilling point. This step has to be done very carefully without breaking the inner membrane of the eggs.

Immediately after the eggs are drilled, the eggs will be transfered to the clean room (Egg Laboratory) for inoculation process through the passbox system. Optimal volume of 0.2ml inoculums is injected per egg. The syringe was thrust down vertically through the hole on the shell at 45° and the inoculum is discharged into the allantoic cavity. The hole is immediately sealed with melted paraffin wax and petroleum jelly to prevent contamination. Then the eggs are incubated for a few days depending on the virus used. After the incubation, the eggs have to be chilled in cold room (+4^oC) for 24 hours in order to harden the blood vessel. This step must be done because it can make the process harvesting easier.

The harvesting step is to collect the allantoic fluid of the eggs. The eggs first have to be swabbed with iodine containing alcohol around the sealed tiny hole in order to further sterilize the outer surface of the eggs. Then, the egg’s shell is broken with forceps according to the air sack marking. The allantoic will be harvested into the sterilized bottle of 500ml. 10ml of the antibiotic will be added in the bottle and 3ml allantoic fluid from each bottle will be taken out for sterility test. All the bottles will then send to the cold room at +4^oC.

BIOTECHNOLOGY LEARNING CURVES SERIES: Introduction to Vaccines Production (Part 4)

Viral Vaccine: Candling & Marking of Eggs

The process for the production of viral vaccine and bacterial vaccine are very different due to the nature of the microbes used.Viral vaccine which is based on viruses needs viable cells (living cells) to survive and propagate meanwhile bacterial vaccine needs only growth nutrient to grow on. Hence, in producing viral vaccine, we need to have incubated eggs (usually SPF- Specific Pathogen Free). SPF means that the eggs are free from 26 common diseases and pathogens. One of the main step in preparing the eggs for vaccine production is eggs candling.

On 7^th day, the eggs will undergo the candling process where the eggs are being observed under the light. The infertile or dead eggs are discarded and the fertile eggs are put in to the incubator for another 3days. The eggs can be differentiated under the light based on the characteristics as follows:

Infertile egg : no embryo

Dead egg : contain embryo but no blood vessel

Fertile egg : contain both embryo and blood vessel

On 10^th day, the eggs will go through the marking process. The eggs are observed under the lamp and the staff will mark the air sack, drilling point and the position of the eye of the embryo. This is an important procedure as it helps in the drilling, inoculation and harvesting procedures later. The marked drilling point indicates the position on the egg shell to be drilled and inoculated while the air sack marked will indicate the position of the shell that can be discarded during the harvesting process. The eye marking are to avoid the inoculation of the virus onto the eye of the embyo which will eventually cause hemorrhagic behavior in the embryo and cause the allantoic fluid to become reddish.

BIOTECHNOLOGY LEARNING CURVES SERIES: Introduction to Vaccines Production (Part 3)

Safety Garments Worn In the Production Plant

The staffs are required to wear proper attire in the plant as an adherence to the GMP standards. In the plants itself, all staffs have to put on laboratory coat, cap, laboratory pants, and laboratory shoes before getting into the production line. Before getting into the clean rooms, the staffs are required to wear special ‘jump-suits’ which include face-mask, hood, garment, and bootie as an extra protective attire to the labcoats, pants, caps and shoes. This is again to avoid contamination to the products during manufacturing processes and to ensure safety of the staffs as they handle bio-hazardous materials.

The figures above show the standard step-by-step procedures in putting on the jumpsuits before getting into the clean rooms: The garments are worn in the changing room which is located in between the clean room and the production line so that all staffs must get changed before getting in from the production line.

BIOTECHNOLOGY LEARNING CURVES SERIES: Introduction to Vaccines Production (Part 2)

GMP Facilities in the Production Plant...

Malaysian Vaccines and Pharmaceuticals is the only GMP certified vaccine prooduction plant in the country. Many facilities are installed in the killed vaccine production plant in Bangi according to the GMP standards. In this section, I will discuss about some of GMP facilities installed and the GMP procedures undergone by all staffs in the plant.

a) Clean Room System

There are 3 clean rooms in the Killed Vaccine Plant in Bangi. They are namely Egg Lab, Tissue Culture Lab and also Filling Lab. The Egg Lab is used for inoculation and harvesting purposes meanwhile tissue cultures lab is used for inactivation process and Filling Lab used for filling and emulsification processes.

The inside of the rooms are pressurized (higher pressure than outside) to hinder air from the outside from going into the rooms. This is to avoid air-borne contamination into the products. Before any production process being conducted in the clean rooms, they have to be fumigated with potassium permanganate and formalin to sterilize to room. This is to maintain the aseptic condition in the laboratories.

b) Passbox System

The equipments and materials used during the production processes in the clean rooms have to be transferred through a special passage which is called the ‘Passbox System’. This is also to ensure aseptic condition being maintained in the process and reduce risks of contamination. Equipments and materials will be put into the passbox from the outside and transferred to the inside of the clean rooms. Before the door from the inside of the rooms can be opened, the equipments or materials will have to go through a blow of laminar flow air. All clean rooms are interconnected through these passboxes. In the egg laboratory, there is another special passbox which is used to transfer bio-hazardous waste such as harvested eggs to the outside of the production plant. The bio-hazardous waste will be transferred from the passbox to the external autoclaves for waste sterilization.

c)Autoclaves

The functions of the autoclaves are pretty obvious; that is to sterilize using heat, all equipments and materials used in the production line as well as waste from production processes. This type of sterilization is called thermal sterilization. The autoclaves operate at high temperature and pressure. There are 2 autoclaves used in MVP, an internal one for sterilization of equipments and materials and an external one for waste disposal.

d) Cold Rooms & Incubators

Cold rooms play a very vital role as well in the vaccines production plant as most of the materials and by-products involved in the production have to be stored at very low temperature. Most of these materials are proteinous substances and cannot stand high temperature, (i.e over 40° C). Some of the materials like the seed virus and bacteria also need to be chilled so that they will remain inactive and maintain the titre (potency) of the virus and bacteria. Some of the common materials stored in the cold room are as follow: \

· Seed Virus and Bacteria: -30°C

· Bulk Virus and Bacteria: +4°C

· Emulsified and inactivated Virus: +4°C

· Alum-Precipitated BacterialVaccines: +4°C

· Vaccines Product: +4°C

There are two cold rooms in the roduction plant which operate at +4°C and another cold box which operates at -30°C. The following figures show the cold rooms used in the plant.

Besides the cold rooms, another equipment used extensively in temperature control is the incubators. Incubators are also called chillers as they perform dual-functions. Incubators are basically a room in which the temperature can be manipulate to suit different roles. Whe the temperature is maintained at high temperature such as +37°C, it become an incubator. This is usually for the purpose of eggs incubation. If the temperature is maintain at low temperature like +20°C, it will become a chiller. The temperature are manipulated by the control panels in the prouction plant.

BIOTECHNOLOGY LEARNING CURVES SERIES: Introduction to Vaccines Production (Part 1)

Introducing Vaccines...

This is a series of posts that are long overdue. In this series, I wish to share what I have learnt in the area of Bioprocess Engineering to all readers. In the first sharing of this series, I would like to share my experience on vaccine production during my tenure as an intern in Malaysian Vaccine and Pharmaceuticals.

So what is vaccine? Vaccine is an antigen used to establish immunity towards a certain disease. So the main difference between drugs and vaccines is vaccine PREVENTS disease before it occurs meanwhile drugs CURE the disease after it occurs.

There are two types of vaccines, which are known as killed and live vaccine. Killed vaccine contains previously virulent microbes which have been killed by heat or chemicals, while live vaccine contains viable, attenuated microbes. The vaccines are also classified based on the micro-organism used, literally viral vaccine which means vaccine from virus and bacterial vaccine which means vaccine from bacteria.